Reperfusion is associated with the induction of several important pro- inflammatory genes, through the activation and nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). The goal of this Project is to characterize the relationships between generation of reactive oxygen species (ROS) following ischemia/reperfusion, the activation of NfkappaB and AP-1, and the expression of a key endothelial cell adhesion molecule intercellular adhesion molecule- (ICAM-1). We will characterize the sources of ROS, including pathways dependent on the small GTP-binding protein Rac-1, responsible for NfkappaB and AP-1 activation in human endothelial cells following hypoxia/reoxygenation. Replication-deficient adenovirus will be used to introduce genes for dominant negative and constitutively active Rac-1 isoforms, ROS generation will be measured by lucigenin and dichlorodihydrofluorescein- based assays, and NfkappaB and AP-1 activation will be assessed by electrophoretic mobility shift assay (EMSA) and transcriptional activity using reporter gene constructs. Effects on cell expression of ICAM-1 and cell apoptosis/death will be assessed. (1) video-microscopy of the ischemic/reperfused cremaster muscle of rats or genetically engineered mice to visualize the microvasculature and measure PMN rolling, adhesion, and extravasation, and (2) the intact canine heart subjected to ischemia/reperfusion. The roles of nitric oxide, performed of newly synthesized tumor necrosis factor alpha, and activated complement will also be examined. The effects of inhibiting NfkappaB following ischemia/reperfusion will be studied vis-a-vis microvascular PMN trafficking in the cremaster preparation and infarct size and myocyte apoptosis in the intact canine and rat heart. The long-term safety of such treatment will also be assessed. The proposed studies should provide important information about the role of ROS and transcription factors NfkappaB and AP-1 in triggering and/or sustaining post-ischemic myocardial inflammation and should lead to the development of new methods to inhibit the inflammatory response and related myocardial injury.